Multiplexed, single-molecule, epigenetic analysis of plasma-isolated nucleosomes for cancer diagnostics.

The analysis of cell-free DNA (cfDNA) in plasma provides information on pathological processes in the body. Blood cfDNA is in the form of nucleosomes, which maintain their tissue- and cancer-specific epigenetic state. We developed a single-molecule multiparametric assay to comprehensively profile the epigenetics of plasma-isolated nucleosomes (EPINUC), DNA methylation and cancer-specific protein biomarkers. Our system allows for high-resolution detection of six active and repressive histone modifications and their ratios and combinatorial patterns on millions of individual nucleosomes by single-molecule imaging. In addition, our system provides sensitive and quantitative data on plasma proteins, including detection of non-secreted tumor-specific proteins, such as mutant p53. EPINUC analysis of a cohort of 63 colorectal cancer, 10 pancreatic cancer and 33 healthy plasma samples detected cancer with high accuracy and sensitivity, even at early stages. Finally, combining EPINUC with direct single-molecule DNA sequencing revealed the tissue of origin of colorectal, pancreatic, lung and breast tumors. EPINUC provides multilayered information of potential clinical relevance from limited (<1 ml) liquid biopsy material.

PRC2-independent actions of H3.3K27M in embryonic stem cell differentiation.

The histone H3 variant, H3.3, is localized at specific regions in the genome, especially promoters and active enhancers, and has been shown to play important roles in development. A lysine to methionine substitution in position 27 (H3.3K27M) is a main cause of Diffuse Intrinsic Pontine Glioma (specifically Diffuse Midline Glioma, K27M-mutant), a lethal type of pediatric cancer. H3.3K27M has a dominant-negative effect by inhibiting the Polycomb Repressor Complex 2 (PRC2) activity. Here, we studied the immediate, genome-wide, consequences of the H3.3K27M mutation independent of PRC2 activity. We developed Doxycycline (Dox)-inducible mouse embryonic stem cells (ESCs) carrying a single extra copy of WT-H3.3, H3.3K27M and H3.3K27L, all fused to HA. We performed RNA-Seq and ChIP-Seq at different times following Dox induction in undifferentiated and differentiated ESCs. We find increased binding of H3.3 around transcription start sites in cells expressing both H3.3K27M and H3.3K27L compared with WT, but not in cells treated with PRC2 inhibitors. Differentiated cells carrying either H3.3K27M or H3.3K27L retain expression of ESC-active genes, in expense of expression of genes related to neuronal differentiation. Taken together, our data suggest that a modifiable H3.3K27 is required for proper histone incorporation and cellular maturation, independent of PRC2 activity.

Lower Extremity Amputee Outcomes with Reference to Co-morbidities.

BACKGROUND: Lower extremity amputation related to diabetes is a serious outcome, which can have devastating effects on the patient and family. The epidemiology of amputations has recently been used as a possible measure of the adequacy of medical prevention and treatment of diabetes and diabetic foot complications. OBJECTIVES: To report on patients undergoing amputations at one medical center in Israel, their co-morbidities, and the outcomes. METHODS: A retrospective chart study was conducted of amputees operated between 1 September 2017 and 30 September 2018. RESULTS: The study population comprised 72 patients who had major amputations for diabetes and/or ischemia, mean age 72 ± 10 years, 74% males, 93% with type 2 diabetes. Mean age corrected Charlson Comorbidity Index was 8.2 ± 2.1 with 90% (65 patients) presenting with a score of 6 or higher. Before the recent deterioration, fewer than 20% of the patients exited their home routinely and 24% had an official diagnosis of dementia. There were 31 below knee amputations (BKA) and 41 above knee amputations (AKA). The 30-day, 3-month, 1-year, and 2-year mortality rates were 15.3%, 27.8%, 43.1%, and 54.2% respectively. Median survival period was 20 months. Survival after AKA was 13.4 ± 20, which was significantly less than after BKA (25.4 ± 2.7, P = 0.097). CONCLUSIONS: Factors other than the quality of management of patients with diabetes and complications may contribute to amputation rates; thus, making speculations from international comparisons of raw amputation rates problematic. This population was less healthy than reported in most studies.

Single-cell epigenetic analysis reveals principles of chromatin states in H3.3-K27M gliomas.

Cancer cells are highly heterogeneous at the transcriptional level and epigenetic state. Methods to study epigenetic heterogeneity are limited in throughput and information obtained per cell. Here, we adapted cytometry by time-of-flight (CyTOF) to analyze a wide panel of histone modifications in primary tumor-derived lines of diffused intrinsic pontine glioma (DIPG). DIPG is a lethal glioma, driven by a histone H3 lysine 27 mutation (H3-K27M). We identified two epigenetically distinct subpopulations in DIPG, reflecting inherent heterogeneity in expression of the mutant histone. These two subpopulations are robust across tumor lines derived from different patients and show differential proliferation capacity and expression of stem cell and differentiation markers. Moreover, we demonstrate the use of these high-dimensional data to elucidate potential interactions between histone modifications and epigenetic alterations during the cell cycle. Our work establishes new concepts for the analysis of epigenetic heterogeneity in cancer that could be applied to diverse biological systems.

Positron charge sensing using a double-gated graphene field effect transistor.

We utilize a high-mobility double-gated graphene field-effect transistor to measure the accumulated charge created by positron annihilation in its back-gate. The device consists of an exfoliated graphene flake stacked between two hexagonal boron nitride flakes placed on a 1 cm2 substrate of 500 µm thick conducting p-doped Si capped by 285 nm-thick SiO2. The device is placed in close proximity to a 780 kBq 22Na positron source emitting a constant flux of positrons. During the measurement, positrons annihilate within the back-gate, kept floating using a low-capacitance relay. The accumulated positive charge capacitively couples to the graphene device and builds a positive voltage, detectable through a shift in the top-gate dependent graphene resistance characteristic. The shift in the position of the top-gate Dirac peak is then used for extracting the exact voltage buildup and quantitative evaluation of the accumulated charge. Reaching a positron current sensitivity of ∼1.2 fA/Hz, detected over 20 min, our results demonstrate the utility of two-dimensional layered materials as probes for charging dynamics of positrons in solids.

Mini Invasive Floating Metatarsal Osteotomy for Diabetic Foot Ulcers Under the First Metatarsal Head: A Case Series.

Diabetic foot ulcers under the first metatarsal head are difficult to treat and prevent recurrence. The aim of this study is to summarize the results of a distal first metatarsal minimally invasive floating osteotomy for ulcers under the first metatarsal head in patients with diabetic neuropathy. We reviewed files of patients with diabetic neuropathy undergoing a floating first metatarsal osteotomy. Demographic and clinical data were collected and analyzed to determine success and complications. We found records for 21 patients (mean age 64) with University of Texas 1A ulcers. The ulcer's mean age was 11.2 months. Following surgery, the ulcer completely resolved after a mean of 3.7 (2 to 11) weeks in 19 patients. During the first year, there were 4 complications related to the surgery (including 3 infections). At latest follow-up, 17/21 (81%) patients had healed with satisfactory results. Minimal invasive floating distal osteotomy of the first metatarsal can cure and prevent recurrence of diabetic foot ulcers under the first metatarsal head in 80% of the patients, but the ability to provide close follow-up and prompt response are prerequisites.

Extra Peritoneal Packing for Exsanguinating Pelvic Hemorrhage: Should We Do It in the Emergency Department?

BACKGROUND: Extra peritoneal packing (EPP) is a quick and highly effective method to control pelvic hemorrhage. OBJECTIVES: To determine whether EPP can be as safely and efficiently performed in the emergency department (ED) as in the operating room (OR). METHODS: Retrospective study of 29 patients who underwent EPP in the ED or OR in two trauma centers in Israel 2008-2018. RESULTS: Our study included 29 patients, 13 in the ED-EPP group and 16 in the OR-EPP group. The mean injury severity score (ISS) was 34.9 ± 11.8. Following EPP, hemodynamic stability was successfully achieved in 25 of 29 patients (86.2%). A raise in the mean arterial pressure (MAP) with a median of 25 mmHg (mean 30.0 ± 27.5, P < 0.001) was documented. All patients who did not achieve hemodynamic stability after EPP had multiple sources of bleeding or fatal head injury and eventually succumbed. Patients who underwent EPP in the ED showed higher change in MAP (P = 0.0458). The overall mortality rate was 27.5% (8/29) with no difference between the OR and ED-EPP. No differences were found between ED and OR-EPP in the amount of transfused blood products, surgical site infections, and length of stay in the hospital. However, patients who underwent ED-EPP were more prone to develop deep vein thrombosis (DVT): 50% (5/10) vs. 9% (1/11) in ED and OR-EPP groups respectively (P = 0.038). CONCLUSIONS: EPP is equally effective when performed in the ED or OR with similar surgical site infection rates but higher incidence of DVT.

Surgical offloading procedures for diabetic foot ulcers compared to best non-surgical treatment: a study protocol for a randomized controlled trial.

BACKGROUND: Diabetic foot ulcers are frequently related to elevated pressure under a bony prominence. Conservative treatment includes offloading with orthopaedic shoes and custom made orthotics or plaster casts. While casting in plaster is usually effective in achieving primary closure of foot ulcers, recurrence rates are high. Minimally invasive surgical offloading that includes correction of foot deformities has good short and long term results. The surgery alleviates the pressure under the bony prominence, thus enabling prompt ulcer healing, negating the patient's dependence on expensive shoes and orthotics, with a lower chance of recurrence. The purpose of this protocol is to compare offloading surgery (percutaneous flexor tenotomy, mini-invasive floating metatarsal osteotomy or Keller arthroplasty) to non-surgical treatment for patients with diabetic foot ulcers in a semi-crossover designed RCT. METHODS: One hundred patients with diabetic neuropathy related foot ulcers (tip of toe ulcers, ulcers under metatarsal heads and ulcers under the hallux interphalangeal joint) will be randomized (2:3) to a surgical offloading procedure or best available non-surgical treatment. Group 1 (surgery) will have surgery within 1 week. Group 2 (controls) will be prescribed an offloading cast applied for up to 12 weeks (based on clinical considerations). Following successful offloading treatment (ulcer closure with complete epithelization) patients will be prescribed orthopaedic shoes and custom made orthotics. If offloading by cast for at least 6 weeks fails, or the ulcer recurs, patients will be offered surgical offloading. Follow-up will take place till 2 years following randomization. Outcome criteria will be time to healing of the primary ulcer (complete epithelization), time to healing of surgical wound, recurrence of ulcer, time to recurrence and complications. DISCUSSION: The high recurrence rate of foot ulcers and their dire consequences justify attempts to find better solutions than the non-surgical options available at present. To promote surgery, RCT level evidence of efficacy is necessary. TRIAL REGISTRATION: Israel MOH_2017-08-10_000719. NIH: NCT03414216.

Extra-peritoneal pressure packing without external pelvic fixation: A life-saving stand-alone surgical treatment.

PURPOSE: Traditional maneuvers aim to decrease retroperitoneal bleeding in hemodynamically unstable multi-trauma patients with unstable pelvic fractures, are reportedly successful in approximately only 50%. The life-saving effect of extra-peritoneal pressure packing (EPPP) is based on direct compression and control of both venous and arterial retroperitoneal bleeders. This study describes the safety and efficacy of emergent EPPP employment, as a stand-alone surgical treatment, that is, carried out without external pelvic fixation or emergent angiography. MATERIALS AND METHODS: A retrospective chart review of all hemodynamic unstable, multi-trauma patients with mechanically unstable pelvic fractures treated by the EPPP technique at our medical center between the years 2005 and 2011. Survival rates, clinical, and physiological outcomes were followed prospectively. RESULTS: Twenty-five of the 181 pelvic fracture patients had biomechanically unstable fractures that required surgical fixation. Fourteen of those 25 patients had deteriorating hemodynamic instability from massive pelvic bleeding which was resistant to resuscitation, and they underwent EPPP, as a stand-alone treatment. The procedure successfully achieved hemodynamic stability in all 14 patients and obviated the early mortality associated with massive pelvic bleeding. Three of these patients eventually succumbed to their multiple injuries. CONCLUSION: Implementation of EPPP improved all measured physiological outcome parameters and survival rates of hemodynamically unstable multi-trauma patients with unstable pelvic fractures in our trauma center. It provided the unique advantage of directly compressing the life-threatening retroperitoneal bleeders by applying direct pressure and causing a tamponade effect to stanch venous and arterial pelvic blood flow and obviate the early mortality associated with massive pelvic bleeding.

Investigation of different magnetic field configurations using an electrical, modular Zeeman slower.

We present a method of constructing an automatically reconfigurable, modular, electronic Zeeman slower, which is remotely controlled. This setup is used to investigate the ability of different magnetic field profiles to slow thermal atoms to the capture velocity of a magneto-optical-trap. We show that a simple numerical optimization process yields better results than the commonly used approach for deciding on the appropriate field and comes close to the optimum field, found by utilizing a fast feedback loop which uses a genetic algorithm. Our new numerical method is easily adaptable to a variety of existing slower designs and may be beneficial where feedback is unavailable.